During the 2011 funding period, we addressed the following: 1) We studied the effects of chronic dietary lithium treatment on robust behavioral and morphological phenotypes in Fmr1 KO mice and found that lithium treatment reversed behavioral and mitigated morphological phenotypes (Liu Z-H et al. Int J Neuropsychopharm 14:618, 2011). Further studies of lithium treatment indicate that lithium also reversed the increased rCPS found in Fmr1 KO mice. A manuscript of the results of rCPS studies is currently under review. Taken together our studies indicate that lithium treatment has a wide range of effects in Fmr1 KO mice suggesting that lithium treatment may affect underlying chemical pathology in FXS. These results in mice suggest that chronic lithium treatment may have therapeutic value in FXS and coupled with the results from other laboratories studying FXS make a strong case for instituting a placebo-controlled trial of lithium in subjects with FXS. 2) Clinical reports suggest that FXS may involve a dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. We have extended our studies of the HPA axis in FXS by investigating the effects of chronic restraint stress in WT and Fmr1 KO mice. We found that mice of both genotypes underwent adrenal hypertrophy and had similar hormonal responses, but only WT mice demonstrated the increased anxiety characteristic of rodents exposed to chronic stress. Moreover only WT mice had morphological changes in dendrites in basolateral amygdala. These differences in response to chronic stress may reflect a diminished adaptive response in Fmr1 KO mice. Results of these studies were published (Qin M et al. Neuroscience (1 September 2011) doi:10.1016/j.neuroscience.2011.06.047). 3) We have characterized a knock-in (KI) mouse model of the fragile X premutation developed by K. Usdin (NIDDK). Our results showed that KI mice have a behavioral phenotype similar to that found in Fmr1 KO mice. We also found clear neuropathology in the KI mouse. Moreover, rCPS were generally higher in KI mice compared with WT. Consistent with findings in the human FX premutation, the level of Fmr1 mRNA was increased, and FMRP levels were reduced. It is thought that symptoms of patients with the FX premutation are due to the FMR1 mRNA toxicity in brain and the age-dependent increase in inclusion bodies. Results of our study highlight similarities in phenotype between KI and Fmr1 KO mice and suggest that it may be the decreased concentration of FMRP that contributes to the phenotype in young adult KI mice. A manuscript reporting these results was published (Qin M et al. Neurobiology of Disease (8 January 2011) doi:10.1016/j.nbd.2011.01.008). A mouse model of the FX premutation developed by R. Willemsen (Erasmus University) is also currently under study in the laboratory. We have confirmed that FMRP levels are about 50% of WT, and we are measuring rCPS in these animals to determine if the effects on rCPS are dependent on the concentration of FMRP. 4) We continued to recruit subjects with FXS to our study of the effects of a lack of FMRP on rCPS. We measured rCPS with the L-1-C-11-leucine PET method in an additional three subjects with FXS and four controls. One of the subjects with FXS was studied under propofol anesthesia;other subjects were studied under dexmedetomidine, a sedating agent that acts by a totally different mechanism. Our preliminary results suggest that, in subjects under propofol sedation, rCPS are not elevated in FXS. In fact, in some brain regions rCPS may be lower in subjects with FXS compared to controls. Propofol acts to potentiate GABA-A receptors and in doing so may modulate some of the effects of glutamate in brain. The response to glutamate is thought to be excessive in FXS. A manuscript of these findings is in preparation. 5) In collaboration with our colleagues from the Walter Reed Army Institute of Research (T. Balkin and D. Picchioni, L-1-C-11-leucine PET studies of the effects of sleep on rCPS in human subjects were initiated this year. We have successfully completed eight studies in which each healthy volunteer was studied three times: awake, sleep-deprived, and during slow wave sleep. Results are being analyzed. 6) We have completed studies of the effects of prior sleep deprivation on the ability of healthy volunteers to learn a TDT. This study is a prelude to our PET study of sleep and memory consolidation in which subjects will undergo training after sleep deprivation followed by a L-1-C-11leucine PET scan while they sleep to ascertain whether rCPS changes in the trained hemisphere. Before we can test the effects on rCPS we showed that subjects could learn under the conditions of prior sleep deprivation. Nine sleep-deprived and nine sleep-sated subjects were administered the TDT before and after a 90 min nap. Both groups showed improved performance after the nap and the improvement was not statistically significantly different between the groups. Our results indicate that prior sleep deprivation does not impede encoding on this perceptual learning task. These results will be presented at the 2011 Meeting of the Society for Neuroscience. 7) Complex mathematical analyses of PET data are successively refined to reduce bias and variability of the estimates of rCPS. We have developed new approaches for the analysis of L-1-C-11-leucine PET data that are more robust to the effects of the limited spatial resolution of the PET camera and noise in the data. To address the impact of kinetic heterogeneity within tissue regions of interest (ROIs) on estimates of rCPS, we developed a method to analyze PET data at the voxel level;we found that voxel-level estimates of rCPS averaged over a ROI were substantially less biased than estimates based on direct fitting of the ROI time-activity curve (TAC) with a homogeneous tissue model (Tomasi G et al, J Cerebr Blood Flow &Metab. 29:1317, 2009). Model fits of the TACs showed that by analyzing the data at the voxel level effects of tissue heterogeneity were reduced, but not entirely eliminated. We also developed an approach that explicitly takes into account heterogeneity within a ROI, spectral analysis with an iterative filter (SAIF). When optimized for and applied to ROI-level data, SAIF produced low bias, low variance estimates of rCPS (Veronese M et al. J Cerebr Blood Flow &Metab. 30, 1460, 2010). rCPS estimated with SAIF does require an assumed constraint on the relationship among the kinetic parameters within the heterogeneous tissue;this has the most impact when kinetics of the various tissues within the ROI are most dissimilar. Reducing the size of a ROI could potentially reduce the kinetic dissimilarity among the tissues within the region and thereby the impact of the kinetic parameter constraint. We have now extended the SAIF method and optimized it for analysis of voxel-level data. rCPS estimated with SAIF was approximately 5-15% higher with voxel-level than ROI-level analysis;intersubject variability was comparable. Simulation studies suggest that the difference is predominantly due to underestimation of rCPS with the ROI-level analysis. A manuscript of these findings is in preparation.